Serum free cultivation of primate embryonic stem cells

ABSTRACT

Disclosed herein are methods for culturing primate embryonic stem cells. These cells are cultured on a prolonged and stable basis in the presence of exogenously supplied fibroblast growth factor and in the absence of animal serum. Preferably there is also a fibroblast feeder layer. Also disclosed is a culture media containing fibroblast feeder layer and the fibroblast growth factor.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH BACKGROUND OF THEINVENTION

[0002] The present invention relates to methods for culturing primateembryonic stem cell cultures and culture media useful therewith.

[0003] Primate (e.g. monkey and human) pluripotent embryonic stem cellshave been derived from preimplantation embryos. See U.S. Pat. No.5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). Thedisclosure of these publications and of all other publications referredto herein are incorporated by reference as if fully set forth herein.Notwithstanding prolonged culture, these cells stably maintain adevelopmental potential to form advanced derivatives of all threeembryonic germ layers.

[0004] Primate (particularly human) ES cell lines have widespreadutility in connection with human developmental biology, drug discovery,drug testing, and transplantation medicine. For example, currentknowledge of the post-implantation human embryo is largely based on alimited number of static histological sections. Because of ethicalconsiderations the underlying mechanisms that control the developmentaldecisions of the early human embryo remain essentially unexplored.

[0005] Although the mouse is the mainstay of experimental mammaliandevelopmental biology, and although many of the fundamental mechanismsthat control development are conserved between mice and humans, thereare significant differences between early mouse and human development.Primate/human ES cells should therefore provide important new insightsinto their differentiation and function.

[0006] Differentiated derivatives of primate ES cells could be used toidentify gene targets for new drugs, used to test toxicity orteratogenicy of new compounds, and used for transplantation to replacecell populations in disease. Potential conditions that might be treatedby the transplantation of ES cell-derived cells include Parkinson'sdisease, cardiac infarcts, juvenile-onset diabetes mellitus, andleukemia. See e.g. J. Rossant et al. 17 Nature Biotechnology 23-4 (1999)and J. Gearhart, 282 Science 1061-2 (1998).

[0007] Long term proliferative capacity, developmental potential afterprolonged culture, and karyotypic stability are key features withrespect to the utility of primate embryonic stem cell cultures. Culturesof such cells (especially on fibroblast feeder layers) have typicallybeen supplemented with animal serum (especially fetal bovine serum) topermit the desired proliferation during such culturing.

[0008] For example, in U.S. Pat. Nos. 5,453,357, 5,670,372 and 5,690,296various culture conditions were described, including some using a typeof basic fibroblast growth factor together with animal serum.Unfortunately, serum tends to have variable properties from batch tobatch, thus affecting culture characteristics.

[0009] In WO 98/30679 there was a discussion of providing a serum-freesupplement in replacement for animal serum to support the growth ofcertain embryonic stem cells in culture. The serum replacement includedalbumins or albumin substitutes, one or more amino acids, one or morevitamins, one or more transferrins or transferrin substitutes, one ormore antioxidants, one or more insulins or insulin substitutes, one ormore collagen precursors, and one or more trace elements. It was notedthat this replacement could be further supplemented with leukemiainhibitory factor, steel factor, or ciliary neurotrophic factor.Unfortunately, in the context of primate embryonic stem cell cultures(especially those grown on fibroblast feeder layers), these culturemedia did not prove satisfactory.

[0010] In the context of nutrient serum culture media (e.g. fetal bovineserum), WO 99/20741 discusses the benefit of use of various growthfactors such as bFGF in culturing primate stem cells. However, culturemedia without nutrient serum is not described.

[0011] In U.S. Pat. No. 5,405,772 growth medium for hematopoietic cellsand bone marrow stromal cells are described. There is a suggestion touse fibroblast growth factor in a serum-deprived media for this purpose.However, conditions for growth primate of embryonic stem cells are notdescribed.

[0012] It can therefore be seen that a need still exists for techniquesto stably culture primate embryonic stem cells without the requirementfor use of animal serum.

BRIEF SUMMARY OF THE INVENTION

[0013] In one aspect the invention provides a method of culturingprimate embryonic stem cells. One cultures the stem cells in a cultureessentially free of mammalian fetal serum (preferably also essentiallyfree of any animal serum) and in the presence of fibroblast growthfactor that is supplied from a source other than just a fibroblastfeeder layer. In a preferred form the culture also has a fibroblastfeeder layer.

[0014] Fibroblast growth factors are essential molecules for mammaliandevelopment. There are currently nine known fibroblast growth factorligands and four signaling fibroblast growth factor receptors therefor(and their spliced variants). See generally D. Ornitz et al., 25 J.Biol. Chem. 15292-7 (1996); U.S. Pat. No. 5,453,357. Slight variationsin these factors are expected to exist between species, and thus theterm fibroblast growth factor is not species limited. However, I preferto use human fibroblast growth factors, more preferably human basicfibroblast growth factor produced from a recombinant gene. This compoundis readily available in quantity from Gibco BRL-Life Technologies andothers.

[0015] It should be noted that for purposes of this patent the culturemay still be essentially free of the specified serum even though adiscrete component (e.g. bovine serum albumin) has been isolated fromserum and then is exogenously supplied. The point is that when serumitself is added the variability concerns arise. However, when one ormore well defined purified component(s) of such serum is added, they donot.

[0016] Preferably the primate embryonic stem cells that are culturedusing this method are human embryonic stem cells that are true ES celllines in that they: (i) are capable of indefinite proliferation in vitroin an undifferentiated state; (ii) are capable of differentiation toderivatives of all three embryonic germ layers (endoderm, mesoderm, andectoderm) even after prolonged culture; and (iii) maintain a normalkaryotype throughout prolonged culture. They are therefore referred toas being pluripotent.

[0017] The culturing permits the embryonic stem cells to stablyproliferate in culture for over one month (preferably over six months;even more preferably over twelve months) while maintaining the potentialof the stem cells to differentiate into derivatives of endoderm,mesoderm, and ectoderm tissues, and while maintaining the karyotype ofthe stem cells.

[0018] In another aspect the invention provides another method ofculturing primate embryonic stem cells. One cultures the stem cells in aculture essentially free of mammalian fetal serum (preferably alsoessentially free of any animal serum) and in the presence of a growthfactor capable of activating a fibroblast growth factor signalingreceptor, wherein the growth factor is supplied from a source other thanjust a fibroblast feeder layer. While the growth factor is preferably afibroblast growth factor, it might also be other materials such ascertain synthetic small peptides (e.g. produced by recombinant DNAvariants or mutants) designed to activate fibroblast growth factorreceptors. See generally T. Yamaguchi et al., 152 Dev. Biol. 75-88(1992)(signaling receptors).

[0019] In yet another aspect the invention provides a culture system forculturing primate embryonic stem cells. It has a fibroblast feeder layerand human basic fibroblast growth factor supplied by other than just thefibroblast feeder layer. The culture system is essentially free ofanimal serum.

[0020] Yet another aspect of the invention provides cell lines(preferably cloned cell lines) derived using the above method. “Derived”is used in its broadest sense to cover directly or indirectly derivedlines.

[0021] Variability in results due to differences in batches of animalserum is thereby avoided. Further, it has been discovered that avoidinguse of animal serum while using fibroblast growth factor can increasethe efficiency of cloning.

[0022] It is therefore an advantage of the present invention to provideculture conditions for primate embryonic stem cell lines where theconditions are less variable and permit more efficient cloning. Otheradvantages of the present invention will become apparent after study ofthe specification and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In the following experiments I used the methods and culturesystems of the invention to culture human ES cell lines. Two clonallyderived human ES cell lines proliferated for over eight months afterclonal derivation and maintained the ability to differentiate toadvanced derivatives of all three embryonic germ layers.

[0024] Techniques for the initial derivation, culture, andcharacterization of the human ES cell line H9 were described in J.Thomson et al., 282 Science 1145-1147 (1998). In my experiments hereinhuman ES cells were then plated on irradiated (35 gray gammairradiation) mouse embryonic fibroblast. Culture medium for the presentwork consisted of 80% “KnockOut” Dulbeco's modified Eagle's medium(DMEM) (Gibco BRL, Rockville, Md.), 1 mM L-Glutamine, 0.1 mMβ-mercaptoethanol, and 1% nonessential amino acids stock (Gibco BRL,Rockville, Md.), supplemented with either 20% fetal bovine serum(HyClone, Logan, Utah) or 20% KnockOut SR, a serum-free replacementoriginally optimized for mouse ES cells (Gibco BRL, Rockville, Md.). Thecomponents of KnockOut SR are those described for serum replacements inWO 98/30679.

[0025] In alternative experiments medium was supplemented with eitherserum or the aforesaid serum replacer KnockOut SR, and either with orwithout human recombinant basic fibroblast growth factor (bFGF, 4ng/ml). The preferred concentration range of bFGF in the culture isbetween 0.1 ng/ml to 500 ng/ml.

[0026] To determine cloning efficiency under varying culture conditions,H-9 cultures were dissociated to single cells for 7 minutes with 0.05%trypsin/0.25% EDTA, washed by centrifugation, and plated on mitoticallyinactivated mouse embryonic fibroblasts (10⁵ ES cells per well of a6-well plate). To confirm growth from single cells for the derivation ofclonal ES cell lines, individual cells were selected by directobservation under a stereomicroscope and transferred by micropipette toindividual wells of a 96 well plate containing mouse embryonicfibroblasts feeders with medium containing 20% serum replacer and 4ng/ml bFGF.

[0027] Clones were expanded by routine passage every 5-7 days with 1mg/ml collagenase type IV (Gibco BRL, Rockville, Md.). Six months afterderivation, H9 cells exhibited a normal XX karyotype by standardG-banding techniques (20 chromosomal spreads analyzed). However, sevenmonths after derivation, in a single karyotype preparation, 16/20chromosomal spreads exhibited a normal XX karyotype, but 4/20 spreadsdemonstrated random abnormalities, including one with a translocation tochromosome 13 short arm, one with an inverted chromosome 20, one with atranslocation to the number 4 short arm, and one with multiplefragmentation. Subsequently, at 8, 10, and 12.75 months afterderivation, H9 cells exhibited normal karyotypes in all 20 chromosomalspreads examined.

[0028] We observed that the cloning efficiency of human ES cells inpreviously described culture conditions that included animal serum waspoor (regardless of the presence or absence of bFGF). We also observedthat in the absence of animal serum the cloning efficiency increased,and increased even more with bFGF.

[0029] The data expressed below is the total number of coloniesresulting from 10⁵ individualized ES cells plated, +/− standard error ofthe mean (percent colony cloning efficiency). With 20% fetal serum andno bFGF there was a result of 240 +/−28. With 20% serum and bFGF theresult was about the same, 260 +/−12. In the absence of the serum(presence of 20% serum replacer) the result with no bFGF was 633 +/−43and the result with bFGF was 826 +/−61. Thus, serum adversely affectedcloning efficiency, and the presence of the bFGF in the absence of serumhad an added synergistic benefit insofar as cloning efficiency.

[0030] The long term culture of human ES cells in the presence of serumdoes not require the addition of exogenously supplied bFGF, and (asnoted above) the addition of bFGF to serum-containing medium does notsignificantly increase human ES cell cloning efficiency. However, inserum-free medium, bFGF increased the initial cloning efficiency ofhuman ES cells.

[0031] Further, I have discovered that supplying exogenous bFGF is veryimportant for continued undifferentiated proliferation of primateembryonic stem cells in the absence of animal serum. In serum-freemedium lacking exogenous bFGF, human ES cells uniformly differentiatedby two weeks of culture. Addition of other factors such as LIF (in theabsence of bFGF) did not prevent the differentiation.

[0032] The results perceived are particularly applicable to clonallines. In this regard, clones for expansion were selected by placingcells individually into wells of a 96 well plate under directmicroscopic observation of 192 H-9 cells plated into wells of 96 wellplates, two clones were successfully expanded (H-9.1 and H-9.2). Both ofthese clones were subsequently cultured continuously in mediasupplemented with serum replacer and bFGF.

[0033] H9.1 and H9.2 cells both maintained a normal XX karyotype evenafter more than 8 months of continuous culture after cloning. The H-9.1and H-9.2 clones maintained the potential to form derivatives of allthree embryonic germ layers even after long term culture in serum-freemedium. After 6 months of culture, H9.1 and H9.2 clones were confirmedto have normal karyotypes and were then injected into SCID-beige mice.

[0034] Both H9.1 and H9.2 cells formed teratomas that containedderivatives of all three embryonic germ layers including gut epithelium(endoderm) embryonic kidney, striated muscle, smooth muscle, bone,cartilage (mesoderm), and neural tissue (ectoderm). The range ofdifferentiation observed within the teratomas of the high passage H9.1and H9.2 cells was comparable to that observed in teratomas formed bylow passage parental H9 cells.

[0035] It should be appreciated from the description above that whileanimal serum is supportive of growth it is a complex mixture that cancontain compounds both beneficial and detrimental to human ES cellculture. Moreover, different serum batches vary widely in their abilityto support vigorous undifferentiated proliferation of human ES cells.Replacing serum with a clearly defined component reduces the variabilityof results associated with this serum batch variation, and should allowmore carefully defined differentiation studies.

[0036] Further, the lower cloning efficiency in medium containing serumsuggests the presence of compounds in conventionally used serum that aredetrimental to stem cell survival, particularly when the cells aredispersed to single cells. Avoiding the use of these compounds istherefore highly desired.

[0037] The present invention has been described above with respect toits preferred embodiments. Other forms of this concept are also intendedto be within the scope of the claims. For example, while recombinantlyproduced human basic fibroblast growth factor was used in the aboveexperiments, naturally isolated fibroblast growth factor should also besuitable. Further, these techniques should also prove suitable for useon monkey and other primate cell cultures.

[0038] Thus, the claims should be looked to in order to judge the fullscope of the invention.

INDUSTRIAL APPLICABILITY

[0039] The present invention provides methods for culturing primateembryonic stem cells, and culture media for use therewith.

I claim:
 1. A method of culturing primate embryonic stem cells,comprising: culturing the stem cells in a culture essentially free ofmammalian fetal serum and in the presence of fibroblast growth factorthat is supplied from a source other than just a fibroblast feederlayer.
 2. The method of claim 1, wherein the culture is essentially freeof any animal serum.
 3. The method of claim 2, wherein the culture alsocomprises a fibroblast feeder layer. 4 The method of claim 2, whereinthe fibroblast growth factor is basic fibroblast growth factor.
 5. Themethod of claim 4, wherein the fibroblast growth factor is human basicfibroblast growth factor which has been produced from a recombinantgene.
 6. The method of claim 2, wherein the primate embryonic stem cellsare human embryonic stem cells.
 7. The method of claim 2, wherein saidculturing step includes the embryonic stem cells proliferating inculture for over one month while maintaining the potential of the stemcells to differentiate into derivatives of endoderm, mesoderm, andectoderm tissues, and while maintaining the karyotype of the stem cells.8. The method of claim 2, wherein the human basic fibroblast growthfactor is present in the culture in a concentration of at least 0.1ng/ml for at least a portion of the method.
 9. A method of culturingprimate embryonic stem cells, comprising: culturing the stem cells in aculture essentially free of mammalian fetal serum and in the presence ofa growth factor capable of activating a fibroblast growth factorsignaling receptor, wherein the growth factor is supplied from a sourceother than just a fibroblast feeder layer.
 10. The method of claim 9,wherein the culture is essentially free of any animal serum.
 11. Themethod of claim 10, wherein the culture also comprises a fibroblastfeeder layer.
 12. The method of claim 10, wherein the primate embryonicstem cells are human embryonic stem cells.
 13. The method of claim 10,wherein said culturing step includes the embryonic stem cellsproliferating in culture for over one month while maintaining thepotential of the stem cells to differentiate into derivatives ofendoderm, mesoderm, and ectoderm tissues, and while maintaining thekatyotype of the stem cells.
 14. A culture system for culturing primateembryonic stem cells, comprising: a fibroblast feeder layer; andfibroblast growth factor supplied by other than just the fibroblastlayer; wherein the culture system is essentially free of animal serum.15. A cell line derived using the method of claim
 1. 16. A cell linederived using the method of claim 9.